Lactuca sativa cultivar CVX-10M Lucky

ABSTRACT

According to the disclosure, there is provided a novel lettuce cultivar, designated Lucky. Lucky is described as a lettuce cultivar with dark green thick leaves, large head size, strong tolerance to tip burn, short core length and round heads. This disclosure thus relates to the seeds of lettuce cultivar Lucky, to the plants of lettuce cultivar Lucky, to plant parts of lettuce cultivar Lucky, to methods for producing a lettuce cultivar by crossing the lettuce cultivar Lucky with another lettuce cultivar, and to methods for producing a lettuce cultivar containing in its genetic material one or more backcross conversion traits or transgenes and to the backcross conversion lettuce plants and plant parts produced by those methods.

CROSS REFERENCE TO RELATED APPLICATION

This is a Continuation Application of U.S. Ser. No. 16/742,578, filedJan. 14, 2020, which is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present disclosure relates to the field of plant breeding. Inparticular, this disclosure relates to a new lettuce variety designatedLucky’.

BACKGROUND OF THE INVENTION

Lettuce is an increasingly popular crop. Worldwide lettuce consumptioncontinues to increase. As a result of this demand, there is a continuedneed for new lettuce varieties. In particular, there is a need forimproved lettuce varieties that exhibit increased resistance to tipburn, corky root rot (CRR), and tomato bushy stunt virus (TBSV) as wellas increased plant uniformity, medium green leaf color, and thicker leaftexture. The present disclosure relates to a new and distinctive icebergor crisphead lettuce (Lactuca sativa L.) variety designated ‘Lucky’. Allpublications cited in this application are herein incorporated byreference.

Most cultivated forms of lettuce belong to the highly polymorphicspecies Lactuca sativa that is grown for its edible head and leaves.Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae)family. Lettuce is related to chicory, sunflower, aster, dandelion,artichoke and chrysanthemum. Sativa is one of about 300 species in thegenus Lactuca.

Presently, there are over a thousand known lettuce varieties withinseven different morphological types. The crisp head group includes theiceberg and batavian types. Iceberg lettuce has a large, firm head witha crisp texture and a white or creamy yellow interior. The batavianlettuce predates the iceberg type and has a smaller and less firm head.The butterhead group has a small, soft head with an almost oily texture.The romaine, also known as cos lettuce, has elongated upright leavesforming a loose, loaf-shaped head and the outer leaves are usually darkgreen. Leaf lettuce comes in many varieties, none of which form a head,and include the green oak leaf variety. The next three types are seldomseen in the United States: Latin lettuce looks like a cross betweenromaine and butterhead; stem lettuce has long, narrow leaves and thick,edible stems; and oilseed lettuce is a type grown for its large seedsthat are pressed to obtain oil.

Lettuce in general and leaf lettuce in particular is an important andvaluable vegetable crop. Thus, a continuing goal of lettuce plantbreeders is to develop stable, high yielding lettuce cultivars that areagronomically sound. To accomplish this goal, the lettuce breeder mustselect and develop lettuce plants with traits that result in superiorcultivars.

Problems with existing cultivars adapted to western conditions include alack of resistance to corky root rot. Corky root rot is believed to becaused by a pathogenic soil bacterium of the genus Rhizomonas. Onespecies of Rhizomonas that is commonly found to cause corky root rot isR. suberifaciens. Corky root rot accounts for significant lettuce croploss in the western United States, particularly in the valleys of thecentral coast of California, i.e., the Salinas, Santa Maria, and Lompocvalleys.

Corky root rot symptoms include yellow bands on tap and lateral roots oflettuce seedlings. Guide to Leafy Vegetable Production in the Far West,Ron Smith, ed., California-Arizona Farm Press (1997). Yellow areasgradually expand and develop a green-brown color with cracks and roughareas on the root surface. The entire taproot may become brown, severelycracked and may cease to function. Feeder root systems are reduced anddamaged. Roots become very brittle and break off easily. When the rootis severely discolored, above ground symptoms show up as wilting duringwarm temperatures, stunting and general poor, uneven growth. Loss of theroot system results in stunted plants that are chlorotic and too smallto harvest.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding preferably begins with the analysis anddefinition of problems and weaknesses of the current germplasm, theestablishment of program goals, and the definition of specific breedingobjectives. The next step is preferably selection of germplasm thatpossess the traits to meet the program goals. The goal is to combine ina single variety or hybrid an improved combination of desirable traitsfrom the parental germplasm.

For a further understanding of lettuce, its uses and history see Waycottet al, U.S. Pat. No. 5,973,232 and Subbarao, K. V (1998) “Progresstowards integrated management of lettuce drop” Plant Dis. 82:1068-1078,which are hereby incorporated by reference in their entirety.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

There is provided a novel crisp head lettuce cultivar, designated Luckythat is particularly adapted to the growing region of the Central Coastof California. This disclosure thus relates to the seeds of lettucecultivar Lucky, to the plants of lettuce cultivar Lucky, to plant partsof lettuce cultivar Lucky, to methods for producing a lettuce cultivarproduced by crossing the lettuce cultivar Lucky with another lettucecultivar, and to methods for producing a lettuce cultivar containing inits genetic material one or more backcross conversion traits ortransgenes and to the backcross conversion lettuce plants and plantparts produced by those methods. This disclosure also relates to lettucecultivars and plant parts derived from lettuce cultivar Lucky, tomethods for producing other lettuce cultivars derived from lettucecultivar Lucky and to the lettuce cultivars and their parts derivedusing those methods. This disclosure further relates to lettuce cultivarseeds, plants and plant parts produced by crossing the lettuce cultivarLucky or a backcross conversion of Lucky with another lettuce cultivar.

There is provided herein a novel lettuce cultivar designated Lucky. Alsoprovided are lettuce plants having the physiological and morphologicalcharacteristics of lettuce cultivar Lucky. This disclosure thus relatesto the seeds of lettuce cultivar Lucky, to the plants of lettucecultivar Lucky, and to methods for producing a lettuce plant produced bycrossing the lettuce cultivar Lucky with itself or another lettuceplant, to methods for producing a lettuce plant containing in itsgenetic material one or more transgenes, and to the transgenic lettuceplants produced by that method. This disclosure also relates to methodsfor producing other lettuce cultivars derived from lettuce cultivarLucky and to the lettuce cultivar derived by the use of those methods.This disclosure further relates to hybrid lettuce seeds and plantsproduced by crossing lettuce cultivar Lucky with another lettucevariety.

In another aspect, the present disclosure provides regenerable cells foruse in tissue culture of lettuce cultivar Lucky. The tissue culture willpreferably be capable of regenerating plants having essentially all ofthe physiological and morphological characteristics of the foregoinglettuce plant, and of regenerating plants having substantially the samegenotype as the foregoing lettuce plant. Preferably, the regenerablecells in such tissue cultures will be callus, protoplasts, meristematiccells, cotyledons, hypocotyl, leaves, pollen, embryos, roots, root tips,anthers, pistils, shoots, stems, petiole flowers, stalks and seeds.Still further, the present disclosure provides lettuce plantsregenerated from the tissue cultures disclosed herein.

The disclosure also relates to methods for producing a lettuce plantcontaining in its genetic material one or more transgenes and to thetransgenic lettuce plant produced by those methods.

Another aspect of the current disclosure is a lettuce plant furthercomprising a single locus conversion. In one embodiment, the lettuceplant is defined as comprising the single locus conversion and otherwisecapable of expressing all of the morphological and physiologicalcharacteristics of the lettuce cultivar Lucky. In particularembodiments, the single locus conversion may comprise a transgenic genewhich has been introduced by genetic transformation into the lettucecultivar Lucky or a progenitor thereof. A transgenic or non-transgenicsingle locus conversion can also be introduced by backcrossing, as iswell known in the art. In still other embodiments of the disclosure, thesingle locus conversion may comprise a dominant or recessive allele. Thelocus conversion may confer potentially any trait upon the single locusconverted plant, including herbicide resistance, insect or pestresistance, resistance to bacterial, fungal, or viral disease, modifiedfatty acid metabolism, modified carbohydrate metabolism, male fertilityor sterility, improved nutritional quality, and industrial usage. Thetrait may be, for example, conferred by a naturally occurring geneintroduced into the genome of the cultivar by backcrossing, a natural orinduced mutation, or a transgene introduced through genetictransformation techniques into the plant or a progenitor of any previousgeneration thereof. When introduced through transformation, a geneticlocus may comprise one or more transgenes integrated at a singlechromosomal location.

The disclosure further relates to methods for genetically modifying alettuce plant of the lettuce cultivar Lucky and to the modified lettuceplant produced by those methods. The genetic modification methods mayinclude, but are not limited to mutation, genome editing, RNAinterference, gene silencing, backcross conversion, genetictransformation, single and multiple gene conversion, and/or direct genetransfer. The disclosure further relates to a genetically modifiedlettuce plant produced by the above methods, wherein the geneticallymodified lettuce plant comprises the genetic modification and otherwisecomprises all of the physiological and morphological characteristics oflettuce cultivar Lucky.

In still yet another aspect, the genetic complement of the lettucecultivar Lucky is provided. The phrase “genetic complement” is used torefer to the aggregate of nucleotide sequences, the expression of whichsequences defines the phenotype of, in the present case, a lettuceplant, or a cell or tissue of that plant. A genetic complement thusrepresents the genetic makeup of a cell, tissue or plant, and a hybridgenetic complement represents the genetic makeup of a hybrid cell,tissue or plant. The disclosure thus provides lettuce plant cells thathave a genetic complement in accordance with the lettuce plant cellsdisclosed herein, and plants, seeds and plants containing such cells.Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.

In still yet another aspect, the disclosure provides a method ofdetermining the genotype of a plant of lettuce cultivar Lucky comprisingdetecting in the genome of the plant at least a first polymorphism. Themethod may, in certain embodiments, comprise detecting a plurality ofpolymorphisms in the genome of the plant. The method may furthercomprise storing the results of the step of detecting the plurality ofpolymorphisms on a computer readable medium. The disclosure furtherprovides a computer readable medium produced by such a method.

This disclosure further relates to the F₁ hybrid lettuce plants andplant parts grown from the hybrid seed produced by crossing lettucecultivar Lucky to a second lettuce plant. Still further included in thedisclosure are the seeds of an F₁ hybrid plant produced with the lettucecultivar Lucky as one parent, the second generation (F₂) hybrid lettuceplant grown from the seed of the F₁ hybrid plant, and the seeds of theF₂ hybrid plant. Thus, any such methods using the lettuce cultivar Luckyare part of this disclosure: selfing, backcrosses, hybrid production,crosses to populations, and the like. All plants produced using lettucecultivar Lucky as at least one parent are within the scope of thisdisclosure. Advantageously, the lettuce cultivar could be used incrosses with other, different, lettuce plants to produce firstgeneration (F₁) lettuce hybrid seeds and plants with superiorcharacteristics.

The disclosure further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding but not limited to recurrent selection, backcrossing, pedigreebreeding, restriction fragment length polymorphism enhanced selection,genetic marker enhanced selection, and transformation. Seeds, lettuceplants, and parts thereof, produced by such breeding methods are alsopart of the disclosure.

This disclosure also relates to lettuce plants or breeding cultivars andplant parts derived from lettuce cultivar Lucky. Still yet anotheraspect of the disclosure is a method of producing a lettuce plantderived from the lettuce cultivar Lucky, the method comprising the stepsof: (a) preparing a progeny plant derived from lettuce cultivar Lucky bycrossing a plant of the lettuce cultivar Lucky with a second lettuceplant; and (b) crossing the progeny plant with itself or a second plantto produce a seed of a progeny plant of a subsequent generation which isderived from a plant of the lettuce cultivar Lucky. In furtherembodiments of the disclosure, the method may additionally comprise: (c)growing a progeny plant of a subsequent generation from said seed of aprogeny plant of a subsequent generation and crossing the progeny plantof a subsequent generation with itself or a second plant; and repeatingthe steps for an additional 2-10 generations to produce a lettuce plantderived from the lettuce cultivar Lucky. The plant derived from lettucecultivar Lucky may be an inbred line, and the aforementioned repeatedcrossing steps may be defined as comprising sufficient inbreeding toproduce the inbred line. In the method, it may be desirable to selectparticular plants resulting from step (c) for continued crossingaccording to steps (b) and (c). By selecting plants having one or moredesirable traits, a plant derived from lettuce cultivar Lucky isobtained which possesses some of the desirable traits of the line aswell as potentially other selected traits. Also provided by thedisclosure is a plant produced by this and the other methods of thedisclosure.

In another embodiment, the method of producing a lettuce plant derivedfrom the lettuce cultivar Lucky further comprises: (a) crossing thelettuce cultivar Lucky-derived lettuce plant with itself or anotherlettuce plant to yield additional lettuce cultivar Lucky-derived progenylettuce seed; (b) growing the progeny lettuce seed of step (a) underplant growth conditions to yield additional lettuce cultivarLucky-derived lettuce plants; and (c) repeating the crossing and growingsteps of (a) and (b) to generate further lettuce cultivar Lucky-derivedlettuce plants. In specific embodiments, steps (a) and (b) may berepeated at least 1, 2, 3, 4, or 5 or more times as desired. Thedisclosure still further provides a lettuce plant produced by this andthe foregoing methods.

The disclosure also provides methods of multiplication or propagation oflettuce plants of the disclosure, which can be accomplished using anymethod known in the art, for example, via vegetative propagation and/orseed. Still further, as another aspect, the disclosure provides a methodof vegetatively propagating a plant of lettuce cultivar Lucky. In anon-limiting example, the method comprises: (a) collecting a plant partcapable of being propagated from a plant of lettuce cultivar Lucky; (b)producing at least a first rooted plant from said plant part. Thedisclosure also encompasses the plantlets and plants produced by thesemethods.

The disclosure further relates to a method of producing a commodityplant product from lettuce cultivar Lucky, such as fresh lettuce leaf,fresh lettuce head, cut, sliced, ground, pureed, dried, canned, jarred,washed, packaged, frozen and/or heated leaves, and to the commodityplant product produced by the method.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of the presentdisclosure, the following definitions are provided:

Allele. The allele is any of one or more alternative forms of a geneticsequence. In a diploid cell or organism, the two alleles of a givengenetic sequence occupy corresponding loci on a pair of homologouschromosomes.

Backcrossing. Backcrossing is a process in which a breeder crossesprogeny back to one of the parents one or more times, for example, afirst-generation hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Bolt. The process during which the stem within the lettuce head greatlyelongates, causing the head to lose its shape and resulting ultimatelyin the producing of a flowering stalk.

Butt. The bottom portion of the lettuce which includes the stem andadjacent leaf bases of the outermost head leaves.

Cell. Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part. The cell can bea cell, such as a somatic cell, of the variety having the same set ofchromosomes as the cells of the deposited seed, or, if the cell containsa locus conversion or transgene, otherwise having the same oressentially the same set of chromosomes as the cells of the depositedseed.

Core. The stem of the lettuce head on which the leaves are borne.

Core Diameter. Diameter of the stem at the base of the cut head.

Core Length. Length of the internal lettuce stem measured from the baseof the cut head to the tip of the core.

Corky root. A disease caused by the bacterium Rhizomonas suberifaciens,which causes the entire taproot to become brown, severely cracked, andnon-functional.

Core Value Coefficient. Calculated by taking the core length andmultiplied by diameter which compares the core shapes. The larger thecore volume coefficient value, the longer and narrower is the core.Inversely, the smaller the core volume coefficient number, the shorterand stubbier the core.

Cotyledon. In the case of lettuce, one of a pair of leaves formed on anembryo within a seed, which upon germination are the first leaves toemerge.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

F_(#) The “F” symbol denotes the filial generation, and the # is thegeneration number, such as F₁, F₂, F₃, etc.

F₁ Hybrid. The first-generation progeny of the cross of two nonisogenicplants.

First outer leaf. As described herein, “first outer leaf” means thefirst leaf located on the outer surface of the lettuce head.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date.

Fourth Leaf. The fourth leaf formed on the lettuce plantlet subsequentto the emergence of the cotyledons.

Frame Diameter. A horizontal measurement of the plant diameter at itswidest point, from outer most leaf tip to outermost leaf tip.

Frame Leaf. The first set of freely recurring leaves which are externalto the head.

Gene. As used herein, “gene” refers to a segment of nucleic acid. A genecan be introduced into a genome of a species, whether from a differentspecies or from the same species, using transformation or variousbreeding methods.

Gene silencing. The interruption or suppression of the expression of agene at the level of transcription or translation.

Genetically modified. Describes an organism that has received geneticmaterial from another organism, or had its genetic material modified,resulting in a change in one or more of its phenotypic characteristics.Methods used to modify, introduce or delete the genetic material mayinclude mutation breeding, genome editing, RNA interference, genesilencing, backcross conversion, genetic transformation, single andmultiple gene conversion, and/or direct gene transfer.

Genome editing. A type of genetic engineering in which DNA is inserted,replaced, modified or removed from a genome using artificiallyengineered nucleases or other targeted changes using homologousrecombination. Examples include but are not limited to use of zincfinger nucleases (ZFNs), TAL effector nucleases (TALENs), meganucleases,CRISPR/Cas9, and other CRISPR related technologies. (Ma et. al.,Molecular Plant, 9:961-974 (2016); Belhaj et. al., Current Opinion inBiotechnology, 32:76-84 (2015)).

Genotype. Refers to the genetic constitution of a cell or organism. Headdiameter. Diameter of the market cut and trimmed head with single capleaf.

Head weight. The weight of a marketable lettuce head, cut and trimmed tomarket specifications.

Leaf area coefficient. Comparison of leaf areas or size between multiplevarieties. This is calculated by multiplying the leaf width by the leaflength.

Leaf Index. Comparison of leaf shape between multiple varieties. This iscalculated by dividing the leaf length by the leaf width.

Linkage. Refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

Linkage disequilibrium. Refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

Locus. A defined segment of DNA.

Locus conversion (also called a ‘trait conversion’ or ‘geneconversion’). A locus conversion refers to a plant or plants within avariety or line that have been modified in a manner that retains theoverall genetics of the variety and further comprises one or more lociwith a specific desired trait, such as but not limited to malesterility, insect or pest control, disease control or herbicidetolerance. Examples of single locus conversions include mutant genes,transgenes and native traits finely mapped to a single locus. One ormore locus conversion traits may be introduced into a single cultivar.

Maturity. Refers to the stage when the plants have mature head formationand are harvestable.

Rogueing. Process in lettuce seed production where undesired plants areremoved from a variety because they differ physically from the general,desired expressed characteristics of the new variety.

Pedigree. Refers to the lineage or genealogical descent of a plant.

Pedigree distance. Relationship among generations based on theirancestral links as evidenced in pedigrees. May be measured by thedistance of the pedigree from a given starting point in the ancestry.

Plant. “Plant” includes plant cells, plant protoplasts, plant tissue,plant cells of tissue culture from which lettuce plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants, or parts of plants such as pollen, flowers, seeds, leaves,stems and the like.

Plant part. Includes any part, organ, tissue or cell of a plantincluding without limitation an embryo, meristem, leaf, pollen,cotyledon, hypocotyl, root, root tip, anther, flower, flower bud,pistil, ovule, seed, shoot, stem, stalk, petiole, pith, capsule, ascion, a rootstock and/or a fruit including callus and protoplastsderived from any of the foregoing.

Plant Cell. As used herein, the term “plant cell” includes plant cellswhether isolated, in tissue culture or incorporated in a plant or plantpart.

Plant Tissue Color Chart. Refers to the Munsell Color Chart for PlantTissue which publishes an official botanical color chart quantitativelyidentifying colors according to a defined numbering system. The MunsellColor Chart for Plant Tissue may be purchased from Munsell ColorServices, 617 Little Britain Road, Suite 102, New Windsor, N.Y.12553-6148, USA, Part Number: 50150.

Quantitative Trait Loci. Quantitative Trait Loci (QTL) refers to geneticloci that control to some degree, numerically representable traits thatare usually continuously distributed.

Single locus converted (conversion) plant. Plants which are developed bya plant breeding technique called backcrossing or via geneticengineering wherein essentially all of the morphological andphysiological characteristics of a variety are recovered in addition tothe desired trait or characteristics conferred by the single locustransferred into the variety via the backcrossing technique or viagenetic engineering. A single locus may comprise one gene, or in thecase of transgenic plants, one or more transgenes integrated into thehost genome at a single site (locus).

Tipburn. Means a browning of the edges or tips of lettuce leaves thathas an unknown cause, possibly a calcium deficiency.

Tomato Bushy Stunt. A disease which causes stunting of growth, leafmottling, and deformed or absent fruit.

Transgene. A nucleic acid of interest that can be introduced into thegenome of a plant by genetic engineering techniques (e.g.,transformation) or breeding.

Lettuce Cultivar Lucky

Crisp head lettuce “Lucky” originated from a manmade cross. The initialcross made in a greenhouse between Central Valley Seeds two proprietaryexperimental lettuce lines the first was used as a female for its sureheading with medium to large size heads, increased tolerance to tip burnand dark green leaf color. The second was used as the male parent forits thicker leaves, smooth flat midribs and higher level of boltingtolerance.

The breeding schemes employed were pedigree selection, using both singleplant selection and mass selection practices. The selection criteria forcv. Lucky was to identify plant types with darker green leaf color(darker than the both parents) with thicker leaf texture, increased headsize for added weight, stronger tolerance to tip burn, shorter corelength with rounder heads. Cv. Lucky displays insignificant to no spiralof the frame leaves.

Replicated field trials for plant selection and breeding were performedin Fresno and Monterey counties. Cultivar Lucky was developed by makingsingle plant sections until the F6 generation. In subsequent years untilthe F9 generation, seeds were massed, and field trialed in differentlocalities.

Cv. Lucky is genetically uniform and stable, and no genetic variants oroff-types have been observed in the last three generations. A varietydescription of Lettuce Cultivar Lucky is provided in Table 1.

TABLE 1 Variety Description Information TRAIT Plant Type Vanguard GroupSEED Color Black (Grey Brown) Light dormancy Light Not Required Heatdormancy Susceptible COTYLEDON TO FOURTH LEAF STAGE Shape of CotyledonsIntermediate Shape of Fourth Leaf Elongated Length/Width Index of FourthLeaf 14 (Length/Width × 10) Apical Margin Crenate/Gnawed Basal MarginModerately Dentate Undulation Slight Green Color Dark Green AnthocyaninDistribution Absent Rolling Absent Cupping Uncupped Reflexing NoneMATURE LEAF Margin Incision Depth Moderate (Vanguard) (deepestpenetration of the margin) Margin Indentation Crenate (Vanguard) (finestdivisions of the margin) Undulations of the Apical Margin Moderate(Vanguard) Green Color Dark green (Vanguard) Anthocyanin DistributionAbsent Size Large Glossiness Glossy (Great Lakes) Blistering Moderate(Vanguard) Leaf Thickness Thick Trichomes Absent (Smooth) PLANT Spreadof Frame Leaves 30 cm Head Diameter 16 cm Head Shape Spherical Head SizeClass Large Head Per Carton 24 Head Weight 842 grams Head Firmnessmoderate BUTT Shape Rounded Midrib Flattened (Salinas) CORE Diameter atBase of Head 38 mm Ration of head diameter/core   04.2 diameter CoreHeight from Base of Head to 49 mm Apex BOLTING Number of Days from FirstWater 49 Date to Seed Stalk Emergence (summer conditions) Bolting ClassMedium Height of Mature Seed Stalk 110 cm Spread of Bolter Plant 41 cmBolter Leaves Curved Margin Dentate Color Dark Green Bolter HabitTerminal Inflorescence Present Bolter Habit Lateral Shoots PresentBolter Habit Basal Side Shoots Present MATURITY Number of Days fromFirst Water 73 Date to Harvest (spring) ADAPTATION Season Summer(Salinas Valley), Fall (Salinas Valley) Soil Type Mineral and OrganicVIRAL DISEASES Big Vein Moderately Resistant/ Moderately SusceptibleFUNGAL/BACTERIAL DISEASES Corky Root Rot Moderately Resistant/Moderately Susceptible Downy Mildew Moderately Resistant/ ModeratelySusceptible Powdery Mildew Moderately Resistant/ Moderately SusceptibleSclerotinia Drop Moderately Resistant/ Moderately SusceptibleVerticillium Wilt Moderately Resistant/ Moderately SusceptiblePHYSIOLOGICAL STRESSES Tipburn Moderately Resistant/ ModeratelySusceptible

Lucky belongs to the iceberg or crisphead lettuce, Lactuca sativa L.varieties. Lucky is described as a vigorous iceberg cultivar andrecommended for the main lettuce growing regions of the Salinas valley.It has darker green leaf color (darker than the both parents) withthicker leaf texture, increased head size for added weight, strongertolerance to tip burn, shorter core length with rounder heads. Cv. Luckydisplays insignificant to no spiral of the frame leaves.

Lucky is most similar to the lettuce variety Tombstone; however, it isdistinct from Tombstone in several characteristics. For example, Luckyhas slight undulation of the fourth leaf and Tombstone has markedundulation; Lucky has dark green color of the fourth leaf whileTombstone has medium green; Lucky has thick glossy leaves, whileTombstone has intermediate thickness and moderately glossy leaves; Luckyhas a spherical heard shape while Tombstone has a slightly flattenedhead shape. Lucky has a rounded butt shape with a flattened midrib,while Tombstone has a flat butt shape with a moderately raised midrib.Most significantly, Lucky has an average head weight of 842 grams whileTombstone has an average head weight of only 670 grams.

Further Embodiments of the Invention

This disclosure is also directed to methods for producing a lettuceplant by crossing a first parent lettuce plant with a second parentlettuce plant, wherein the first parent lettuce plant or second parentlettuce plant is the lettuce plant from cultivar Lucky. Further, boththe first parent lettuce plant and second parent lettuce plant may befrom cultivar Lucky. Therefore, any methods using lettuce cultivar Luckyare part of this disclosure, such as selfing, backcrosses, hybridbreeding, and crosses to populations. Plants produced using lettucecultivar Lucky as at least one parent are within the scope of thisdisclosure.

In one aspect of the disclosure, methods for developing novel planttypes are presented. In one embodiment the specific type of breedingmethod is pedigree selection, where both single plant selection and massselection practices are employed. Pedigree selection, also known as the“Vilmorin system of selection,” is described in Fehr, Walter; Principlesof Cultivar Development, Volume I, Macmillan Publishing Co., which ishereby incorporated by reference.

In lettuce breeding, lines may be selected for certain desiredappropriate characteristics. To optimize crossing, it is important tonote that lettuce is an obligate self-pollinating species. This meansthat the pollen is shed before stigma emergence, assuring 100%self-fertilization. Since each lettuce flower is an aggregate of about10-20 individual florets (typical of the Compositae family), removal ofthe anther tubes containing the pollen is performed by procedures wellknown in the art of lettuce breeding.

In one embodiment, the pedigree method of breeding is practiced whereselection is first practiced among F₂ plants. In the next season, themost desirable F₃ lines are first identified, and then desirable F₃plants within each line are selected. The following season and in allsubsequent generations of inbreeding, the most desirable families areidentified first, then desirable lines within the selected families arechosen, and finally desirable plants within selected lines are harvestedindividually. A family refers to lines that were derived from plantsselected from the same progeny row the preceding generation.

Using this pedigree method, two parents may be crossed using anemasculated female and a pollen donor (male) to produce F₁ offspring. Tooptimize crossing, it is important to note that lettuce is an obligateself-pollinating species. This means that the pollen is shed beforestigma emergence, assuring 100% self-fertilization. Since each lettuceflower is an aggregate of about 10-20 individual florets, manual removalof the anther tubes containing the pollen is tedious. As such, methodsof removing pollen well known to one of skill in the art, such asmisting to wash the pollen off prior to fertilization, may be employedto assure crossing or hybridization. The F₁ may be self-pollinated toproduce a segregating F₂ generation. Individual plants may then beselected which represent the desired phenotype in each generation (F₃,F₄, F₅, etc.) until the traits are homozygous or fixed within a breedingpopulation.

In addition to crossing, selection may be used to identify and isolatenew lettuce lines. In lettuce selection, lettuce seeds are planted, theplants are grown, and single plant selections are made of plants withdesired characteristics. Seed from the single plant selections may beharvested, separated from seeds of the other plants in the field andre-planted. The plants from the selected seed may be monitored todetermine if they exhibit the desired characteristics of the originallyselected line. Selection work is preferably continued over multiplegenerations to increase the uniformity of the new line.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding may be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Each breeding program may include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard, theoverall value of the advanced breeding lines, and the number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

In one embodiment, promising advanced breeding lines are thoroughlytested and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for new commercial cultivars; those still deficient in a fewtraits are used as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, usually take several years from the time the first crossor selection is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of lettuce plant breeding is to develop new, unique andsuperior lettuce cultivars. In one embodiment, the breeder initiallyselects and crosses two or more parental lines, followed by repeatedselfing and selection, producing many new genetic combinations. Thebreeder can theoretically generate billions of different geneticcombinations via crossing, selfing and mutations. Preferably, each yearthe plant breeder selects the germplasm to advance to the nextgeneration. This germplasm may be grown under different geographical,climatic and soil conditions, and further selections are then made,during and at the end of the growing season.

In a preferred embodiment, the development of commercial lettucecultivars requires the development of lettuce varieties, the crossing ofthese varieties, and the evaluation of the crosses. Pedigree breedingand recurrent selection breeding methods may be used to developcultivars from breeding populations. Breeding programs may combinedesirable traits from two or more varieties or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars may becrossed with other varieties and the hybrids from these crosses areevaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are usually selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (e.g., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals may be identified or created byintercrossing several different parents. The best plants may be selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. Preferably, the selected plants are intercrossed toproduce a new population in which further cycles of selection arecontinued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent may beselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure refers to planting a segregatingpopulation, harvesting a sample of one seed per plant, and using theone-seed sample to plant the next generation. When the population hasbeen advanced from the F₂ to the desired level of inbreeding, the plantsfrom which lines are derived will each trace to different F₂individuals. The number of plants in a population declines eachgeneration due to failure of some seeds to germinate or some plants toproduce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, (Molecular Linkage Map ofSoybean (Glycine max) p 6.131-6.138 in S. J. O'Brien (ed) Genetic Maps:Locus Maps of Complex Genomes, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1993)) developed a molecular genetic linkage mapthat consisted of 25 linkage groups with about 365 RFLP, 11 RAPD, threeclassical markers and four isozyme loci. See also, Shoemaker, R. C.,RFLP Map of Soybean, p 299-309, in Phillips, R. L. and Vasil, I. K.,eds. DNA-Based Markers in Plants, Kluwer Academic Press, Dordrecht, theNetherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. (Diwan, N. and Cregan, P.B., Theor. Appl. Genet. 95:22-225, 1997.) SNPs may also be used toidentify the unique genetic composition of the disclosure and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intolettuce varieties. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation (such as X-rays, Gamma rays,neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens(such as base analogs like 5-bromo-uracil), antibiotics, alkylatingagents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Principles of Cultivar Development byFehr, Macmillan Publishing Company, 1993.

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan et al., Theor. Appl. Genet., 77:889-892, 1989.

Targeting Induced Local Lesions in Genomes (TILLING)

Breeding schemes of the present application can include crosses withTILLING® plant cultivars. TILLING® is a method in molecular biology thatallows directed identification of mutations in a specific gene. TILLING®was introduced in 2000, using the model plant Arabidopsis thaliana.TILLING® has since been used as a reverse genetics' method in otherorganisms such as zebrafish, corn, wheat, rice, soybean, tomato andlettuce.

The method combines a standard and efficient technique of mutagenesiswith a chemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with asensitive DNA screening-technique that identifies single base mutations(also called point mutations) in a target gene. EcoTILLING is a methodthat uses TILLING® techniques to look for natural mutations inindividuals, usually for population genetics analysis (see Comai, etal., 2003 The Plant Journal 37, 778-786; Gilchrist et al. 2006 Mol.Ecol. 15, 1367-1378; Mejlhede et al. 2006 Plant Breeding 125, 461-467;Nieto et al. 2007 BMC Plant Biology 7, 34-42, each of which isincorporated by reference hereby for all purposes). DEcoTILLING is amodification of TILLING® and EcoTILLING which uses an inexpensive methodto identify fragments (Garvin et al., 2007, DEco-TILLING: An inexpensivemethod for SNP discovery that reduces ascertainment bias. MolecularEcology Notes 7, 735-746).

The TILLING® method relies on the formation of heteroduplexes that areformed when multiple alleles (which could be from a heterozygote or apool of multiple homozygotes and heterozygotes) are amplified in a PCR,heated, and then slowly cooled. A “bubble” forms at the mismatch of thetwo DNA strands (the induced mutation in TILLING® or the naturalmutation or SNP in EcoTILLING), which is then cleaved by single strandednucleases. The products are then separated by size on several differentplatforms.

Several TILLING® centers exists over the world that focus onagriculturally important species: UC Davis (USA), focusing on Rice;Purdue University (USA), focusing on Maize; University of BritishColumbia (CA), focusing on Brassica napus; John Innes Centre (UK),focusing on Brassica rapa; Fred Hutchinson Cancer Research, focusing onArabidopsis; Southern Illinois University (USA), focusing on Soybean;John Innes Centre (UK), focusing on Lotus and Medicago; and INRA(France), focusing on Pea and Tomato. More detailed description onmethods and compositions on TILLING® can be found in U.S. Pat. No.5,994,075, US 2004/0053236 A1, WO 2005/055704, and WO 2005/048692, eachof which is hereby incorporated by reference for all purposes.

Thus, in some embodiments, the breeding methods of the presentdisclosure include breeding with one or more TILLING plant lines withone or more identified mutations. Descriptions of other breeding methodsthat are commonly used for different traits and crops can be found inone of several reference books (e.g., Principles of Plant Breeding JohnWiley and Son, pp. 115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep etal., 1979; Fehr, 1987; “Carrots and Related Vegetable Umbelliferae”,Rubatzky, V. E., et al., 1999).

Lettuce is an important and valuable vegetable crop. Thus, a continuinggoal of lettuce plant breeders is to develop stable, high yieldinglettuce cultivars that are agronomically sound. To accomplish this goal,the lettuce breeder preferably selects and develops lettuce plants withtraits that result in superior cultivars.

This disclosure also is directed to methods for producing a lettucecultivar plant by crossing a first parent lettuce plant with a secondparent lettuce plant wherein either the first or second parent lettuceplant is a lettuce plant of the line Lucky. Further, both first andsecond parent lettuce plants can come from the cultivar Lucky. Stillfurther, this disclosure also is directed to methods for producing acultivar Lucky-derived lettuce plant by crossing cultivar Lucky with asecond lettuce plant and growing the progeny seed and repeating thecrossing and growing steps with the cultivar Lucky-derived plant from 0to 7 times. Thus, any such methods using the cultivar Lucky are part ofthis disclosure: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cultivar Lucky as aparent are within the scope of this disclosure, including plants derivedfrom cultivar Lucky. Advantageously, the cultivar is used in crosseswith other, different, cultivars to produce first generation (F₁)lettuce seeds and plants with superior characteristics.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which lettuce plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, seeds, roots,anthers, and the like.

As is well known in the art, tissue culture of lettuce can be used forthe in vitro regeneration of a lettuce plant. Tissue culture of varioustissues of lettuces and regeneration of plants therefrom is well knownand widely published. For example, reference may be had to Teng et al.,HortScience. 1992, 27: 9, 1030-1032 Teng et al., HortScience. 1993, 28:6, 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992, 46:3, 287-290, Webb et al., Plant Cell Tissue and Organ Culture. 1994, 38:1, 77-79, Curtis et al., Journal of Experimental Botany. 1994, 45: 279,1441-1449, Nagata et al., Journal for the American Society forHorticultural Science. 2000, 125: 6, 669-672. It is clear from theliterature that the state of the art is such that these methods ofobtaining plants are, and were, “conventional” in the sense that theyare routinely used and have a very high rate of success. Thus, anotheraspect of this disclosure is to provide cells which upon growth anddifferentiation produce lettuce plants having the physiological andmorphological characteristics of variety Lucky.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively astransgenes. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentdisclosure, in particular embodiments, also relates to transformedversions of the claimed line.

Plant transformation preferably involves the construction of anexpression vector that will function in plant cells. Such a vector maycomprise DNA comprising a gene under control of or operatively linked toa regulatory element (for example, a promoter). The expression vectormay contain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid and can beused alone or in combination with other plasmids, to provide transformedlettuce plants, using transformation methods as described below toincorporate transgenes into the genetic material of the lettuceplant(s).

Expression Vectors for Lettuce Transformation

Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galaetesidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teen et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters

Genes included in expression vectors preferably are driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, promoter includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive promoter” is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inlettuce. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce. With an inducible promoter the rateof transcription increases in response to an inducing agent. Anyinducible promoter can be used in the instant disclosure. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter may be operably linked to a gene for expressionin lettuce or the constitutive promoter may be operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce.

Many different constitutive promoters can be utilized in the instantdisclosure. Exemplary constitutive promoters include, but are notlimited to, the promoters from plant viruses such as the 35S promoterfrom CaMV (Odell et al., Nature 313:810-812 (1985) and the promotersfrom such genes as rice actin (McElroy et al., Plant Cell 2:163-171(1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632(1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU(Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten etal., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al.,Mol. Gen. Genetics 231:276-285 (1992) and Atanassova et al., PlantJournal 2 (3): 291-300 (1992)). The ALS promoter, Xba1/Ncol fragment 5′to the Brassica napus ALS3 structural gene (or a nucleotide sequencesimilarity to said Xba1/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT application WO96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter may be operably linked to a gene forexpression in lettuce. Optionally, the tissue-specific promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in lettuce. Plantstransformed with a gene of interest operably linked to a tissue-specificpromoter produce the protein product of the transgene exclusively, orpreferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant disclosure. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondroin or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Additional Methods for Genetic Engineering of Lettuce

In general, methods to transform, modify, edit or alter plant endogenousgenomic DNA include altering the plant native DNA sequence or apre-existing transgenic sequence including regulatory elements, codingand non-coding sequences. These methods can be used, for example, totarget nucleic acids to pre-engineered target recognition sequences inthe genome. Such pre-engineered target sequences may be introduced bygenome editing or modification. As an example, a genetically modifiedplant variety is generated using “custom” or engineered endonucleasessuch as meganucleases produced to modify plant genomes (see e.g., WO2009/114321; Gao et al. (2010) Plant Journal 1:176-187). Anothersite-directed engineering method is through the use of zinc fingerdomain recognition coupled with the restriction properties ofrestriction enzyme. See e.g., Urnov, et al., (2010) Nat Rev Genet.11(9):636-46; Shukla, et al., (2009) Nature 459 (7245):437-41. Atranscription activator-like (TAL) effector-DNA modifying enzyme (TALEor TALEN) is also used to engineer changes in plant genome. See e.g.,US20110145940, Cermak et al., (2011) Nucleic Acids Res. 39(12) and Bochet al., (2009), Science 326(5959): 1509-12. Site-specific modificationof plant genomes can also be performed using the bacterial type IICRISPR (clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated) system and other similar methods. See e.g., Belhajet al., (2013), Plant Methods 9: 39; The Cas9/guide RNA-based systemallows targeted cleavage of genomic DNA guided by a customizable smallnoncoding RNA in plants (see e.g., WO 2015026883A1, incorporated hereinby reference).

A genetic map can be generated that identifies the approximatechromosomal location of an integrated DNA molecule, for example viaconventional restriction fragment length polymorphisms (RFLP),polymerase chain reaction (PCR) analysis, simple sequence repeats (SSR),and single nucleotide polymorphisms (SNP). For exemplary methodologiesin this regard, see Glick and Thompson, Methods in Plant MolecularBiology and Biotechnology, pp. 269-284 (CRC Press, Boca Raton, 1993).

Wang et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome”, Science (1998)280:1077-1082, and similar capabilities are increasingly available forthe lettuce genome. Map information concerning chromosomal location isuseful for proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants to determine if the latter have a commonparentage with the subject plant. Map comparisons could involvehybridizations, RFLP, PCR, SSR, sequencing or combinations thereof, allof which are conventional techniques. SNPs may also be used alone or incombination with other techniques.

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present disclosure, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants that areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is lettuce. In anotherpreferred embodiment, the biomass of interest is seed. For transgenicplants that show higher levels of expression, a genetic map can begenerated, primarily via conventional RFLP, PCR and SSR analysis, whichidentifies the approximate chromosomal location of the integrated DNAmolecule. For exemplary methodologies in this regard, see Glick andThompson, Methods in Plant Molecular Biology and Biotechnology CRCPress, Boca Raton 269:284 (1993). Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants, to determine if the latter have acommon parentage with the subject plant. Map comparisons may involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present disclosure, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotoch. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper accumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung lettuce calmodulin cDNA clones, and Griesset al., Plant Physiol. 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of tachyolesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1, 4-D-galacturonase. See Lamb at al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa lettuce endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2:367 (1992).

R. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bioi/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

S. A lettuce mosaic potyvirus (LMV) coat protein gene introduced intoLactuca sativa in order to increase its resistance to LMV infection. SeeDinant et al., Molecular Breeding. 1997, 3: 1, 75-86.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase PAT bar genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Seealso Umaballava-Mobapathie in Transgenic Research. 1999, 8: 1, 33-44that discloses Lactuca sativa resistant to glufosinate. European patentapplication No. 0 333 033 to Kumada at al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in European application No. 0 242 246 to Leemans et al.,DeGreef et al., Bio/Technology 7:61 (1989), describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. Exemplary of genesconferring resistance to phenoxy propionic acids and cycloshexones, suchas sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genesdescribed by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori et al., Mol. Gen.Genet. 246:419, 1995. Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., PlantPhysiol., 106:17, 1994), genes for glutathione reductase and superoxidedismutase (Aono et al., Plant Cell Physiol. 36:1687, 1995), and genesfor various phosphotransferases (Datta et al., Plant Mol. Biol. 20:619,1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and international publication WO 01/12825.

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Increased iron content of the lettuce, for example by transforming aplant with a soybean ferritin gene as described in Goto et al., ActaHorticulturae. 2000, 521, 101-109. Parallel to the improved iron contentenhanced growth of transgenic lettuces was also observed in earlydevelopment stages.

B. Decreased nitrate content of leaves, for example by transforming alettuce with a gene coding for a nitrate reductase. See for exampleCurtis et al., Plant Cell Report. 1999, 18: 11, 889-896.

C. Increased sweetness of the lettuce by transferring a gene coding formonellin that elicits a flavor sweeter than sugar on a molar basis. SeePenarrubia et al., Biotechnology. 1992, 10: 5, 561-564.

D. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89:2625 (1992).

E. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus licheniformis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Sogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

4. Genes that Control Male-Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622, 1992).

Methods for Lettuce Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). Curtis et al., Journal ofExperimental Botany. 1994, 45: 279, 1441-1449, Torres et al., Plant cellTissue and Organic Culture. 1993, 34: 3, 279-285, Dinant et al.,Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Several methods of plant transformation collectively referred to asdirect gene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 to 4 μm.The expression vector is introduced into plant tissues with a biolisticdevice that accelerates the microprojectiles to speeds of 300 to 600 m/swhich is sufficient to penetrate plant cell walls and membranes.Russell, D. R., et al. Pl. Cell. Rep. 12(3, January), 165-169 (1993),Aragao, F. J. L., et al. Plant Mol. Biol. 20(2, October), 357-359(1992), Aragao, F. J. L., et al. Pl. Cell. Rep. 12(9, July), 483-490(1993). Aragao, Theor. Appl. Genet. 93: 142-150 (1996), Kim, J.;Minamikawa, T. Plant Science 117: 131-138 (1996), Sanford et al., Part.Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988),Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C., PhysiolPlant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion has been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-omithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514(1997/98), Donn et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin etal., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol.24:51-61 (1994). See also Chupean et al., Biotechnology. 1989, 7: 5,503-508.

Following transformation of lettuce target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossed,with another (non-transformed or transformed) line, in order to producea new transgenic lettuce line. Alternatively, a genetic trait that hasbeen engineered into a particular lettuce cultivar using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Gene Conversions

When the term lettuce plant, cultivar or lettuce line is used in thecontext of the present disclosure, this also includes any geneconversions of that line. The term gene converted plant as used hereinrefers to those lettuce plants which are developed by a plant breedingtechnique called backcrossing wherein essentially all of the desiredmorphological and physiological characteristics of a cultivar arerecovered in addition to the gene transferred into the line via thebackcrossing technique. Backcrossing methods can be used with thepresent disclosure to improve or introduce a characteristic into theline. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental lettuce plantsfor that line. The parental lettuce plant that contributes the gene forthe desired characteristic is termed the nonrecurrent or donor parent.This terminology refers to the fact that the nonrecurrent parent is usedone time in the backcross protocol and therefore does not recur. Theparental lettuce plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the originalcultivar of interest (recurrent parent) is crossed to a second line(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until alettuce plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute traits or characteristics in the original line.To accomplish this, a gene or genes of the recurrent cultivar aremodified or substituted with the desired gene or genes from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original line. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross. One of the major purposes is to add some commerciallydesirable, agronomically important trait or traits to the plant. Theexact backcrossing protocol will depend on the characteristics or traitsbeing altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many gene traits have been identified that are not regularly selectedfor in the development of a new line but that can be improved bybackcrossing techniques. Gene traits may or may not be transgenic,examples of these traits include but are not limited to, herbicideresistance, resistance for bacterial, fungal, or viral disease, insectresistance, enhanced nutritional quality, industrial usage, yieldstability, yield enhancement, male sterility, modified fatty acidmetabolism, and modified carbohydrate metabolism. These genes aregenerally inherited through the nucleus. Several of these gene traitsare described in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of lettuce andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng et al., HortScience. 1992, 27: 9,1030-1032 Teng et al., HortScience. 1993, 28: 6, 669-1671, Zhang et al.,Journal of Genetics and Breeding. 1992, 46: 3, 287-290, Webb et al.,Plant Cell Tissue and Organ Culture. 1994, 38: 1, 77-79, Curtis et al.,Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Nagata et al.,Journal for the American Society for Horticultural Science. 2000, 125:6, 669-672, and Ibrahim et al., Plant Cell, Tissue and Organ Culture.(1992), 28(2): 139-145. It is clear from the literature that the stateof the art is such that these methods of obtaining plants are routinelyused and have a very high rate of success. Thus, another aspect of thisdisclosure is to provide cells which upon growth and differentiationproduce lettuce plants having the physiological and morphologicalcharacteristics of cultivar Lucky.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Additional Breeding Methods

This disclosure also is directed to methods for producing a lettuceplant by crossing a first parent lettuce plant with a second parentlettuce plant wherein the first or second parent lettuce plant is alettuce plant of cultivar Lucky. Further, both first and second parentlettuce plants can come from lettuce cultivar Lucky. Thus, any suchmethods using lettuce cultivar Lucky are part of this disclosure:selfing, backcrosses, hybrid production, crosses to populations, and thelike. All plants produced using lettuce cultivar Lucky as at least oneparent are within the scope of this disclosure, including thosedeveloped from cultivars derived from lettuce cultivar Lucky.Advantageously, this lettuce cultivar could be used in crosses withother, different, lettuce plants to produce the first generation (F₁)lettuce hybrid seeds and plants with superior characteristics. Thecultivar of the disclosure can also be used for transformation whereexogenous genes are introduced and expressed by the cultivar of thedisclosure. Genetic variants created either through traditional breedingmethods using lettuce cultivar Lucky or through transformation ofcultivar Lucky by any of a number of protocols known to those of skillin the art are intended to be within the scope of this disclosure.

The following describes breeding methods that may be used with lettucecultivar Lucky in the development of further lettuce plants. One suchembodiment is a method for developing cultivar Lucky progeny lettuceplants in a lettuce plant breeding program comprising: obtaining thelettuce plant, or a part thereof, of cultivar Lucky, utilizing saidplant or plant part as a source of breeding material, and selecting alettuce cultivar Lucky progeny plant with molecular markers in commonwith cultivar Lucky and/or with morphological and/or physiologicalcharacteristics selected from the characteristics listed in Table 1.Breeding steps that may be used in the lettuce plant breeding programinclude pedigree breeding, backcrossing, mutation breeding, andrecurrent selection. In conjunction with these steps, techniques such asRFLP-enhanced selection, genetic marker enhanced selection (for exampleSSR markers) and the making of double haploids may be utilized.

Another method which may be used involves producing a population oflettuce cultivar Lucky-progeny lettuce plants, comprising crossingcultivar Lucky with another lettuce plant, thereby producing apopulation of lettuce plants, which, on average, derive 50% of theiralleles from lettuce cultivar Lucky. A plant of this population may beselected and repeatedly selfed or sibbed with a lettuce cultivarresulting from these successive filial generations. One embodiment ofthis disclosure is the lettuce cultivar produced by this method and thathas obtained at least 50% of its alleles from lettuce cultivar Lucky.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus the disclosure includes lettucecultivar Lucky progeny lettuce plants comprising a combination of atleast two cultivar Lucky traits selected from the group consisting ofthose listed in Table 1 or the cultivar Lucky combination of traitslisted above, so that said progeny lettuce plant is not significantlydifferent for said traits than lettuce cultivar Lucky as determined atthe 5% significance level when grown in the same environmentalconditions. Using techniques described herein, molecular markers may beused to identify said progeny plant as a lettuce cultivar Lucky progenyplant. Mean trait values may be used to determine whether traitdifferences are significant, and preferably the traits are measured onplants grown under the same environmental conditions. Once such avariety is developed its value is substantial since it is important toadvance the germplasm base as a whole in order to maintain or improvetraits such as yield, disease resistance, pest resistance, and plantperformance in extreme environmental conditions.

Progeny of lettuce cultivar Lucky may also be characterized throughtheir filial relationship with lettuce cultivar Lucky, as for example,being within a certain number of breeding crosses of lettuce cultivarLucky. A breeding cross is a cross made to introduce new genetics intothe progeny, and is distinguished from a cross, such as a self or a sibcross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween lettuce cultivar Lucky and its progeny. For example, progenyproduced by the methods described herein may be within 1, 2, 3, 4 or 5breeding crosses of lettuce cultivar Lucky.

The foregoing disclosure has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be obvious that certain changes and modifications suchas single gene modifications and mutations, somoclonal variants, variantindividuals selected from large populations of the plants of the instantvariety and the like may be practiced within the scope of thedisclosure, as limited only by the scope of the appended claims.

Deposits

Applicant(s) have made a deposit of at least 625 seeds of LettuceCultivar Lucky with the American Type Culture Collection (ATCC),Manassas, Va. 20110 USA, ATCC Deposit No. PTA-127244. The seedsdeposited with the ATCC on Jan. 11, 2022 were taken from the depositmaintained by Central Valley Seeds, 485 Victor Way, Suite 10, SalinasCalif. 93907 since prior to the filing date of this application. Accessto this deposit will be available during the pendency of the applicationto the Commissioner of Patents and Trademarks and persons determined bythe Commissioner to be entitled thereto upon request. Upon issue ofclaims, the Applicant(s) will make available to the public, pursuant to37 CFR 1.808, a deposit of at least 625 seeds of cultivar Lucky with theAmerican type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209. This deposit of the lettuce cultivar Luckywill be maintained in the ATCC depository, which is a public depository,for a period of 30 years, or 5 years after the most recent request, orfor the enforceable life of the patent, whichever is longer, and will bereplaced if it becomes nonviable during that period. Additionally,Applicants have or will satisfy all the requirements of 37 C.F.R. §§1.801-1.809, including providing an indication of the viability of thesample. Applicants have no authority to waive any restrictions imposedby law on the transfer of biological material or its transportation incommerce. Applicants do not waive any infringement of their rightsgranted under this patent or under the Plant Variety Protection Act (7USC 2321 et seq.).

What is claimed is:
 1. A seed of lettuce cultivar Lucky, wherein arepresentative sample of seed of said lettuce having been depositedunder ATCC Accession No. PTA-127244.
 2. A lettuce plant, or a partthereof or a plant cell thereof, produced by growing the seed ofclaim
 1. 3. The lettuce part of claim 2, wherein the lettuce part isselected from the group consisting of: a leaf, a flower, a head, anovule, pollen and a cell.
 4. A lettuce plant having all of thephysiological and morphological characteristics of lettuce cultivarLucky when grown in the same environmental conditions, or a part or aplant cell thereof.
 5. A tissue or culture of regenerable cells producedfrom the plant or plant part of claim 2, wherein cells of the tissueculture are produced from a plant part selected from the groupconsisting of protoplasts, embryos, meristematic cells, callus, pollen,ovules, flowers, seeds, leaves, roots, root tips, anthers, stems,petioles, cotyledons and hypocotyls.
 6. A lettuce plant regenerated fromthe tissue culture of claim 5, said plant having the physiological andmorphological characteristics of lettuce cultivar Lucky, wherein arepresentative sample of seed of said lettuce having been depositedunder ATCC Accession No. PTA-127244.
 7. A lettuce head produced from theplant of claim
 2. 8. A method for producing a lettuce head comprising a)growing the lettuce plant of claim 2 to produce a lettuce head, and b)harvesting said lettuce head.
 9. A method for producing a lettuce seedcomprising crossing a first parent lettuce plant with a second parentlettuce plant and harvesting the resultant F₁ lettuce seed, wherein saidfirst parent lettuce plant and/or second parent lettuce plant is thelettuce plant of claim
 2. 10. An F₁ lettuce seed produced by the methodof claim
 9. 11. The method of claim 9, wherein the method furthercomprises: (a) crossing a plant grown from said F₁ lettuce seed withitself or a different lettuce plant to produce a seed of a progeny plantof a subsequent generation; (b) growing a progeny plant of a subsequentgeneration from said seed of a progeny plant of a subsequent generationand crossing the progeny plant of a subsequent generation with itself ora second plant to produce a progeny plant of a further subsequentgeneration; and (c) repeating steps (a) and (b) using said progeny plantof a further subsequent generation from step (b) in place of the plantgrown from said F₁ lettuce seed in step (a), wherein steps (a) and (b)are repeated with sufficient inbreeding to produce an inbred lettuceplant derived from the lettuce cultivar Lucky.
 12. A method forproducing a lettuce seed comprising self-pollinating the lettuce plantof claim 2 and harvesting the resultant lettuce seed.
 13. An F₁ lettuceseed produced by the method of claim
 12. 14. A method of producing alettuce plant derived from the lettuce cultivar Lucky, the methodcomprising the steps of: (a) crossing the plant of claim 2 with a secondlettuce plant to produce a progeny plant; (b) crossing the progeny plantof step (a) with itself or the second lettuce plant in step (a) toproduce a seed; (c) growing a progeny plant of a subsequent generationfrom the seed produced in step (b); (d) crossing the progeny plant of asubsequent generation of step (c) with itself or the second lettuceplant in step (a) to produce a lettuce plant derived from the lettucecultivar Lucky.
 15. The method of claim 14 further comprising the stepof: (e) repeating step b) and/or c) for at least 1 more generation toproduce a lettuce plant derived from the lettuce cultivar Lucky.
 16. Theplant or plant part of claim 2, wherein the plant further comprises atleast one locus conversion and otherwise comprises all of thephysiological and morphological characteristics of the lettuce cultivarLucky.
 17. The plant of claim 16, wherein the locus conversion conferssaid plant with a trait selected from the group consisting of malesterility, male fertility, herbicide resistance, insect resistance,disease resistance, water stress tolerance, heat tolerance, improvedshelf life, delayed shelf life, and improved nutritional quality. 18.The plant of claim 17, wherein the locus conversion is an artificiallymutated gene or nucleotide sequence.
 19. A method of introducing adesired trait into lettuce cultivar Lucky comprising: (a) crossing aplant of lettuce cultivar Lucky grown from seed of lettuce cultivarLucky, wherein a representative sample of seed has been deposited underATCC Accession No. PTA-127244, with another lettuce plant that comprisesa desired trait to produce at least one F₁ progeny plant, wherein thedesired trait is selected from the group consisting of insectresistance, herbicide resistance, disease resistance, water stresstolerance, heat tolerance, improved shelf life, delayed shelf life, andimproved nutritional quality; (b) selecting one or more progeny plantsthat have the desired trait to produce at least one selected progenyplant; (c) crossing the at least one selected progeny plant with atleast one plant of the lettuce cultivar Lucky to produce at least onebackcross progeny plant; (d) selecting for at least one backcrossprogeny plant that has the desired trait and the physiological andmorphological characteristics of lettuce cultivar Lucky when grown inthe same environmental conditions to produce at least one selectedbackcross progeny plant; and (e) repeating steps (c) and (d) three ormore times in succession to produce at least one selected fourth orhigher backcross progeny plant that comprises the desired trait and thephysiological and morphological characteristics of lettuce cultivarLucky when grown in the same environmental conditions.
 20. A method fordeveloping a lettuce plant in a lettuce plant breeding program,comprising applying plant breeding techniques comprising recurrentselection, backcrossing, pedigree breeding, marker enhanced selection,or transformation to the lettuce plant of claim 2, or its parts, whereinapplication of said techniques results in development of a lettuceplant.
 21. A method of vegetatively propagating a plant of lettucecultivar Lucky, wherein the method comprises: (a) collecting a plantpart capable of being propagated from a plant of lettuce cultivar Lucky,wherein a representative sample of seed of said cultivar was depositedunder ATCC Accession No. PTA-127244; and (b) producing at least a firstrooted plantlet or plant from said plant part.
 22. A lettuce plantlet orplant produced by the method of claim 21, wherein the lettuce plantletor plant has all of the physiological and morphological characteristicsof lettuce cultivar Lucky.
 23. A method of producing a geneticallymodified lettuce plant, wherein the method comprises mutation,transformation, gene conversion, genome editing, RNA interference orgene silencing of the plant of claim
 2. 24. A genetically modifiedlettuce plant produced by the method of claim 23, wherein the plantcomprises the genetic modification and otherwise comprises all of thephysiological and morphological characteristics of the lettuce cultivarLucky.
 25. A method of determining a genotype of lettuce cultivar Lucky,or a first-generation progeny thereof, the method comprising: (a)obtaining a sample of nucleic acids from the plant of claim 2; and (b)detecting a polymorphism in the nucleic acid sample.
 26. A method ofproducing a commodity plant product, comprising obtaining the plant ofclaim 2, or a plant part thereof, and producing the commodity plantproduct from said plant or plant part thereof, wherein said commodityplant product is selected from the group consisting of fresh lettuceleaf, fresh lettuce head, cut, sliced, ground, pureed, dried, canned,jarred, washed, packaged, frozen and heated leaves.
 27. A commodityplant product produced by the method of claim 26, wherein the commodityplant product comprises at least one cell of lettuce cultivar Lucky.